Development and characterization of the NanoOrange® protein quantitation assay:: A fluorescence-based assay of proteins in solution
Development and characterization of the NanoOrange® protein quantitation assay:: A fluorescence-based assay of proteins in solution
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DOI：
10.2144/03344pt03
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发表时间：
2003-04-01
期刊：
影响因子：
2.7
通讯作者：
Singer, VL
中科院分区：
文献类型：
--
作者：
Jones, LJ;Haugland, RP;Singer, VL
我们开发了一种灵敏的荧光测定法，使用 NanoOrange(R) 试剂对溶液中的蛋白质进行定量，该试剂是一种部花青染料，在与去污剂包被的蛋白质相互作用后，荧光量子产率会大幅增加。 NanoOrange 测定允许使用标准荧光计检测 10 ng/mL 至 10 mug/mL 蛋白质，提供广泛的动态定量范围，并相对于基于吸收的蛋白质溶液测定提高了灵敏度。 NanoOrange 测定的蛋白质间变异性与标准测定法相当，包括 Lowry、二辛可宁酸和 Bradford 程序。我们还发现 NanoOrange 检测可用于检测相对较小的蛋白质或较大的肽，例如抑肽酶和胰岛素。该测定对蛋白质制剂中发现的几种常见污染物（例如盐和洗涤剂）的存在有些敏感；然而，它对还原剂、核酸和游离氨基酸的存在不敏感。简单的测定方案适合自动化。在含有去污剂的稀释剂中存在染料的情况下，将样品短暂加热，冷却至室温，然后使用 485 mn 激发波长和 590 mn 发射波长测量荧光。因此，NanoOrange 检测非常适合与标准荧光酶标仪、荧光计和一些激光扫描仪一起使用。
We developed a sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange((R)) reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins. The NanoOrange assay allowed for the detection of 10 ng/mL to 10 mug/mL protein with a standard fluorometer, offering a broad, dynamic quantitation range and improved sensitivity relative to absorption-based protein solution assays. The protein-to-protein variability of the NanoOrange assay was comparable to those of standard assays, including Lowry, bicinchoninic acid, and Bradford procedures. We also found that the NanoOrange assay is useful for detecting relatively small proteins or large peptides, such as aprotinin and insulin. The assay was somewhat sensitive to the presence of several common contaminants found in protein preparations such as salts and detergents; however, it was insensitive to the presence of reducing agents, nucleic acids, and free amino acids. The simple assay protocol is suitable for automation. Samples are briefly heated in the presence of dye in a detergent-containing diluent, allowed to cool to room temperature, and fluorescence is measured using 485-mn excitation and 590-mn emission wavelengths. Therefore, the NanoOrange assay is well suited for use with standard fluorescence microplate readers, fluorometers, and some laser scanners.